Combination Therapies Using PRMT5 Inhibitors for the Treatment of Cancer

ABSTRACT

This disclosure relates to methods of treating cancer. This disclosure further relates to treating cancer in a subject with compounds that are inhibitors of PRMT5, particularly in combination with CDK4/6 inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.63/172,643, filed Apr. 8, 2021, and U.S. Provisional Application No.63/253,029, filed Oct. 6, 2021, the disclosure of each of which ishereby incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE Field of the Disclosure

This disclosure relates to methods of treating cancer. This disclosurefurther relates to treating cancer in a subject with compounds that areinhibitors of protein arginine N-methyl transferase 5 (PRMT5),particularly in combination with cyclin-dependent kinase 4 and 6(CDK4/6) inhibitors.

Description of Related Art

PRMT5 is a type II arginine methyltransferase that catalyzes thetransfer of a methyl group from S-adenosyl-L-methionine (SAM) to anomega-nitrogen of the guanidino function of protein L-arginine residues(omega-monomethylation) and the transfer of a second methyl group to theother omega-nitrogen, yielding symmetric dimethylarginine (sDMA). PRMT5forms a complex with methylosome protein 50 (MEP50), which is requiredfor substrate recognition and orientation and is also required forPRMT5-catalyzed histone 2A and histone 4 methyltransferase activity(e.g., see Ho et al. (2013) PLoS ONE 8(2): e57008).

Homozygous deletions of p16/CDKN2a are prevalent in cancer and thesemutations commonly involve the co-deletion of adjacent genes, includingthe gene encoding methylthioadenosine phosphorylase (MTAP). It isestimated that approximately 15% of all human cancers have a homozygousdeletion of the MTAP gene (e.g., see Firestone & Schramm (2017) J. Am.Chem Soc. 139(39):13754-13760).

Cells lacking MTAP activity have elevated levels of the MTAP substrate,methylthioadenosine (MTA), which is a potent inhibitor of PRMT5.Inhibition of PRMT5 activity results in reduced methylation activity andincreased sensitivity of cellular proliferation to PRMT5 depletion orloss of activity. Hence, the loss of MTAP activity reduces methylationactivity of PRMT5 making the cells selectively dependent on PRMT5activity.

Despite importance of PRMT5 on cell viability and its prevalence incancers, effective therapies that inhibit PRMT5 have been elusive. Thus,there remains a need to develop new PRMT5 inhibitor therapies to treatwide range of cancers.

SUMMARY OF THE DISCLOSURE

One aspect of the disclosure provides methods for treating cancer in asubject. Such methods include administering to the subject atherapeutically effective amount of a CDK4/6 inhibitor and atherapeutically effective amount of a PRMT5 inhibitor.

Also provided herein is a method for treating cancer in a subject inneed thereof. Such methods include determining that the cancer isassociated with MTAP homozygous deletion (e.g., an MTAP-associatedcancer). These methods optionally further include determining that thecancer is associated with a CDKN2A homozygous deletion. Such methodsfurther include administering to the subject a therapeutically effectiveamount of a CDK4/6 inhibitor and a therapeutically effective amount of aPRMT5 inhibitor.

These and other features and advantages of the present invention will bemore fully understood from the following detailed description takentogether with the accompanying claims. It is noted that the scope of theclaims is defined by the recitations therein and not by the specificdiscussion of features and advantages set forth in the presentdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the methods of the disclosure, and are incorporated inand constitute a part of this specification. The drawings illustrate oneor more embodiment(s) of the disclosure and, together with thedescription, serve to explain the principles and operation of thedisclosure.

FIG. 1 illustrates the results of the methods of Example 1 in theKRAS^(G12C) and CDKN2A/MTAP^(DEL) lung tumor xenograft LU99 model grownin immunodeficient mice. The PRMT5 inhibitor used in this method wasMRTX9768 administered at 100 mg/kg twice a day (BID), and the CDK4/6inhibitor was palbociclib administered at 130 mg/kg once a day (QD).Average tumor volume±standard error is plotted of the mean at study dayas indicated.

FIG. 2 illustrates the results of the methods of Example 2 in theKRAS^(G12C) and CDKN2A/MTAP^(DEL) lung tumor xenograft LU99 model grownin immunodeficient mice. The PRMT5 inhibitor used in this method wasMRTX7477 administered at 200 mg/kg BID, and the CDK4/6 inhibitor waspalbociclib administered at 130 mg/kg QD. Average tumor volume±standarderror is plotted of the mean at study day as indicated.

FIG. 3 illustrates the results of the methods of Example 3 in HCC4006lung tumor xenograft model. The PRMT5 inhibitor used in this method wasMRTX1719 administered at 100 mg/kg QD, and the CDK4/6 inhibitor waspalbociclib administered at 130 mg/kg QD. Average tumor volume±standarderror is plotted of the mean at study day as indicated.

FIG. 4 illustrates the results of the methods of Example 4 in SW1573PRMT5-044 lung tumor xenograft model. The PRMT5 inhibitor used in thismethod was MRTX1719 administered at 50 mg/kg QD, and the CDK4/6inhibitor was palbociclib administered at 130 mg/kg QD. Average tumorvolume±standard error is plotted of the mean at study day as indicated.

FIG. 5 illustrates the results of the methods of Example 5 in H1650 lungtumor xenograft model. The PRMT5 inhibitor used in this method wasMRTX1719 administered at 100 mg/kg QD, and the CDK4/6 inhibitor waspalbociclib administered at 130 mg/kg QD. Average tumor volume±standarderror is plotted of the mean at study day as indicated.

FIG. 6 illustrates the results of the methods of Example 6 in A549PRMT-034 lung tumor xenograft model. The PRMT5 inhibitor used in thismethod was MRTX1719 administered at 100 mg/kg QD, and the CDK4/6inhibitor was palbociclib administered at 130 mg/kg QD. Average tumorvolume±standard error is plotted of the mean at study day as indicated.

FIG. 7 illustrates the results of the methods of Example 7 in PANC-05-04pancreatic tumor xenograft model. The PRMT5 inhibitor used in thismethod was MRTX1719 administered at 100 mg/kg QD, and the CDK4/6inhibitor was palbociclib administered at 130 mg/kg QD. Average tumorvolume±standard error is plotted of the mean at study day as indicated.

FIG. 8 illustrates the results of the methods of Example 8 in BXPC-3pancreatic tumor xenograft model. The PRMT5 inhibitor used in thismethod was MRTX1719 administered at 100 mg/kg QD, and the CDK4/6inhibitor was palbociclib administered at 130 mg/kg QD. Average tumorvolume±standard error is plotted of the mean at study day as indicated.

FIG. 9 illustrates the results of the methods of Example 9 in MKN45gastric tumor xenograft model. The PRMT5 inhibitor used in this methodwas MRTX1719 administered at 100 mg/kg QD, and the CDK4/6 inhibitor waspalbociclib administered at 130 mg/kg QD. Average tumor volume±standarderror is plotted of the mean at study day as indicated.

DETAILED DESCRIPTION OF THE DISCLOSURE

Before the disclosed processes and materials are described, it is to beunderstood that the aspects described herein are not limited to specificembodiments, and as such can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and, unless specifically definedherein, is not intended to be limiting.

In view of the present disclosure, the methods and compositionsdescribed herein can be configured by the person of ordinary skill inthe art to meet the desired need. The present disclosure providesimprovements in treating cancer in a subject. As used herein, the terms“subject” or “patient” are used interchangeably, refers to any animal,including mammals, and most preferably humans.

The methods provided herein may be used for the treatment of a widevariety of cancer including tumors such as lung, prostate, breast,brain, skin, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to tumor typessuch as astrocytic, breast, cervical, colorectal, endometrial,esophageal, gastric, head and neck, hepatocellular, laryngeal, lung,oral, ovarian, prostate and thyroid carcinomas and sarcomas. Morespecifically, these compounds can be used to treat: Cardiac: sarcoma(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma,rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma(squamous cell, undifferentiated small cell, undifferentiated largecell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor(nephroblastoma), lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Biliary tract: gallbladder carcinoma, ampullary carcinoma, cholangiocarcinoma; Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma (serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic:blood (myeloid leukemia (acute and chronic), acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myelodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma (malignant lymphoma); Skin: malignant melanoma,basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;and Adrenal glands: neuroblastoma.

In certain embodiments of the methods of the disclosure, the cancer is aMTAP-associated cancer. For example, in certain embodiments, the cancercomprises MTAP gene homozygous deletion (MTAP^(DEL)). The subject may beidentified or diagnosed as having MTAP-associated cancer where, forexample, MTAP^(DEL) is determined using a suitable assay or a kit.Alternatively, the subject is suspected of having MTAP-associated canceror the subject has a clinical record indicating that the subject hasMTAP-associated cancer.

In certain embodiments of the methods of the disclosure, the cancercomprises a cyclin-dependent kinase inhibitor 2A (CDKN2A) genehomozygous deletion (CDKN2A^(DEL)). The subject may be identified ordiagnosed as having CDKN2A^(DEL) where the deletion is determined usinga suitable assay or a kit. Alternatively, the subject is suspected ofhaving the CDKN2A^(DEL) cancer, or the subject has a clinical recordindicating that the subject has the CDKN2A^(DEL) cancer.

In certain embodiments of the methods of the disclosure, the cancer mayfurther comprise a Kirsten rat sarcoma viral oncogene homolog (KRAS)gene mutation, such as glycine-to-cysteine (KRAS^(G12C)) gene mutation.The subject may be identified or diagnosed as having KRAS^(G12C) cancerwhere KRAS^(G12C) mutation is determined using a suitable assay or akit. Alternatively, the subject is suspected of having the KRAS^(G12C)cancer or the subject has a clinical record indicating that the subjecthas the KRAS^(G12C) cancer.

In some embodiments of any of the methods or uses described herein, anassay is used to determine whether the patient has MTAP^(DEL) and/orCDKN2A^(DEL) and/or KRAS^(G12C) using a sample (e.g., a biologicalsample or a biopsy sample such as a paraffin-embedded biopsy sample)from a subject. Such assay includes, but is not limited to, nextgeneration sequencing, immunohistochemistry, fluorescence microscopy,break apart FISFI analysis, Southern blotting. Western blotting, FACSanalysis, Northern blotting, and PCR-based amplification (e.g., RT-PCRand quantitative real-time RT-PCR). As is well known in the art, theassays are typically performed, e.g., with at least one labelled nucleicacid probe or at least one labelled antibody or antigen-binding fragmentthereof.

In certain embodiments, the cancer in the methods of the disclosure isselected from lung cancer, pancreatic cancer, head and neck cancer,bladder cancer, esophageal cancer, lymphoma, stomach cancer, skincancer, breast cancer, brain cancer, liver cancer, and colon cancer.

In certain embodiments, the cancer in the methods of the disclosure isselected from lung cancer, pancreatic cancer, head and neck cancer,bladder cancer, esophageal cancer, lymphoma, stomach cancer, skincancer, breast cancer, and brain cancer.

In certain embodiments, the cancer in the methods of the disclosure isselected from lung cancer (e.g., mesothelioma or non-small cell lungcancer (NSCLC) including adenocarcinoma and squamous cell), pancreaticcancer, head and neck cancer (such as squamous cell carcinoma (HNSCC)),bladder cancer, esophageal cancer, lymphoma (e.g., diffuse large B-celllymphoma), stomach cancer, melanoma, breast cancer, and brain cancer(e.g., glioblastoma multiforme and glioma).

In certain embodiments, the cancer in the methods of the disclosure isselected from mesothelioma, NSCLC (e.g., adenocarcinoma and squamouscell), pancreatic cancer, HNSCC, and bladder cancer.

In one embodiment of the methods of the disclosure, the cancer is lungcancer. For example, the lung cancer may be NSCLC (e.g., adenocarcinomaand squamous cell) or mesothelioma.

In one embodiment of the methods of the disclosure, the cancer ispancreatic cancer. In another embodiment, the cancer is head and neckcancer. In yet another embodiment, the cancer is bladder cancer.

As provided above, the CDK4/6 inhibitor is administered in the methodsof the disclosure. As used herein, a “CDK4/6 inhibitor” refers tocompounds capable of negatively modulating or inhibiting all or aportion of the enzymatic activity of CDK4/6. The CDK4/6 inhibitors ofthe present disclosure interact with and/or irreversibly bind to CDK4/6resulting in the inhibition of the enzymatic activity of CDK4/6 andblocking the transition from the G1 to the S phase of the cell cycle.

In certain embodiments, the CDK4/6 inhibitor also inhibits CDK2, i.e.,it is a CDK 2/4/6 inhibitor.

In certain embodiments, the CDK4/6 inhibitor is selected frompalbociclib (sold as Ibrance®, Pfizer Inc., New York, N.Y.), abemaciclib(sold as Verzenio®, Eli Lilly and Company, Indianapolis, Ind.),ribociclib (sold as Kisqali®, Novartis Pharmaceuticals, Basel,Switzerland), PF-06873600 (CAS No. 2185857-97-8, available from PfizerInc., New York, N.Y.), and combinations thereof. In certain embodiments,the CDK4/6 inhibitor is trilaciclib (sold as Cosela™, G1 Therapeutics,Inc., Durham, N.C.).

In one embodiment of the methods of the disclosure, the CDK4/6 inhibitoris palbociclib.

As provided above, the PRMT5 inhibitor is also administered in themethods of the disclosure. A “PRMT5 inhibitor” as used herein refers tocompounds of the disclosure as described herein. These compounds arecapable of negatively modulating or inhibiting all or a portion of theenzymatic activity of the PRMT5, particularly, in the presence of boundMTA in vitro or in vivo or in cells expressing elevated levels of MTA.In certain embodiments, the PRMT5 inhibitor is a MTA-cooperative PRMT5inhibitor.

In certain embodiments, the PRMT5 inhibitor of the disclosure is any oneof the PRMT5 inhibitors disclosed in International patent publicationNo. WO 2021/050915 A1, published 18 Mar. 2021, incorporated by referencein its entirety.

In certain other embodiments, the PRMT5 inhibitor of the disclosure isany one of the PRMT5 inhibitors disclosed in U.S. provisionalapplication No. 63/200,521, filed 11 Mar. 2021, incorporated byreference in its entirety.

For example, the PRMT5 inhibitor in the methods of the disclosure asdescribed herein is a compound of Formula IIA, IIB or IIC (Embodiment1):

or a pharmaceutically acceptable salt thereof, wherein:

A is CR⁹ or N;

D is (C(R⁹)₂)₁₋₂—NH₂,

or D is

where the methylene is bonded to E where E is C;

E is C, CR⁹ or N;

each L is independently a bond or C₁-C₃ alkylene;

W is CR⁹ or N;

each X is independently a bond, O, S, —NR⁴— or —NR⁴C(O)—;each Z is independently a bond, —SO—, —SO₂—, —CH(OH)— or —C(O)—;each R² is independently hydroxy, halogen, cyano, cyanomethyl, —(NR⁴)₂,hydroxyalkyl, alkoxy, —SO₂C₁-C₃alkyl, —X-arC₁-C₃alkyl, heteroalkyl,C₂-C₄ alkynyl, —X-haloalkyl, —X-C₁-C₅ alkyl, —Z-C₁-C₅ alkyl,heterocyclyl, —X-L-cycloalkyl, —Z-cycloalkyl, —X-aryl, —Z-aryl, or—X-heteroaryl, wherein the heterocyclyl, the cycloalkyl, the aryl andthe heteroaryl are optionally substituted with one or more R⁵;each R⁴ is independently hydrogen or C₁-C₃ alkyl;each R⁵ is independently cyano, oxo, halogen, C₁-C₃ alkyl, hydroxyalkyl,hydroxy, alkoxy, alkoxy-C₁-C₃ alkyl, —X-haloalkyl, —Z-cycloalkyl,—X-arC₁-C₃alkyl, —X-arC₁-C₃alkyl substituted with cyano, —X-L-cycloalkyloptionally substituted with C₁-C₃ alkyl or oxo, —X-L-heteroaryloptionally substituted with one or more C₁-C₃ alkyl or oxo,—X-L-heterocyclyl optionally substituted with one or more C₁-C₃ alkyl oroxo, or —X-aryl;R⁶ is hydrogen, halogen, C₁-C₃ alkyl, haloalkyl, hydroxy, alkoxy, C₁-C₃alkyl-alkoxy, N(R⁹)₂, NR⁹C(O)R⁹, C(O)R⁹, oxetane and THF;R⁷ is H or C₁-C₃ alkyl optionally substituted with one or more halogen;R⁸ is H or C₁-C₃ alkyl; andeach R⁹ is independently H or C₁-C₃ alkyl, halogen or haloalkyl.

Embodiment 2 provides the PRMT5 inhibitor in the methods of thedisclosure as a compound of Formula IIA:

Embodiment 3 provides the PRMT5 inhibitor in the methods of thedisclosure as a compound of Formula IIB:

Embodiment 4 provides the PRMT5 inhibitor in the methods of thedisclosure as a compound of Formula IIC:

Embodiment 5 provides the method of any of embodiments 1-4, wherein W isCR⁹.

Embodiment 6 provides the method of any of embodiments 1-4, wherein A isCR⁹.

Embodiment 7 provides the method of any of embodiments 1-4, wherein E isN.

Embodiment 8 provides the method of any of embodiments 1-7, wherein W isCR⁹, A is CR⁹ and E is N.

Embodiment 9 provides the method of any of embodiments 1-8, wherein R²is selected from: benzothiophene, naphthalene, quinoline, chromane,isochromane, dihydrobenzodioxine, indolazine, tetrahydroindolazine,dihydroisobenzofuran, benzene, isoquinolinone, benzodioxone,thienopyridine, tetrahydroindolone, indolizine, dihydroindolizinone,imadazopyridinone, thienopyrimidine, thiophene, pyrrolopyrimidinone,thiazolopyridinone, dihydropyrrolizine, isoindalone andtetrahydroisoquinoline.

Embodiment 10 provides the method of any of embodiments 1-8, whereineach R⁵ is independently cyano, oxo, halogen, C1-C3 alkyl, hydroxy,hydroxyalkyl, alkoxy-C1-C3alkyl, —X-L-heterocyclyl optionallysubstituted with one or more C1-C3alkyl or oxo, —X-L-cycloalkyloptionally substituted with C1-C3 alkyl or oxo.

Embodiment 11 provides the method of any of embodiments 1-8, wherein R⁶is selected from hydrogen, hydroxy, chlorine, —NHC(O)CH₃, —C(O)CF₂H,—NH₂, —CF₂, —CH₃, —O—CH₂CH₃, —CH₂—CH₂—O—CH₃, oxetane and THF.

Embodiment 12 provides the method of any of embodiments 1-11, where oneof L, X and Z is a bond.

Embodiment 13 provides the method of embodiment 12, wherein all of L, Xand Z are bonds.

One aspect of the disclosure provides the method wherein the PRMT5inhibitor is a compound of the formula (IIIC) (Embodiment 14):

or a pharmaceutically acceptable salt thereof, wherein

A is CR⁹ or N;

D is —CH₂—NH₂,

W is CR⁹ or N, where R⁹ is H or C₁-C₃ alkyl;G, Q, J and U are independently selected from C(H), C(R⁵), and N,provided only one or two of G, Q, J, and U can be N;

each R⁵ is independently hydroxy, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₃-C₆ cycloalkoxy, C₃-C₆ cycloalkyl,C₃-C₆ heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl;

R⁶ is hydrogen, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, hydroxy, C₁-C₆alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶, where each R⁹ is independently H orC₁-C₃ alkyl, R¹⁵ is hydrogen or methyl, and R¹⁶ is C₁-C₃ alkyl; andR⁷ is C₁-C₃ alkyl or C₁-C₃ haloalkyl.

Embodiment 15 provides the method according to embodiment 14, wherein Ais CH.

Embodiment 16 provides the method according to embodiment 14 or 15,wherein W is N.

Embodiment 17 provides the method according to embodiment 14 or 15,wherein W is CH.

Embodiment 18 provides the method according to any of embodiments 14-17,wherein D is —CH₂—NH₂.

Embodiment 19 provides the method of the disclosure wherein the PRMT5inhibitor is a compound according to embodiment 14 of the formula:

Embodiment 20 provides the method according to any of embodiments 14-19,wherein R⁶ is hydrogen, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, hydroxy,C₁-C₆ alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl,—C(O)—C₁-C₃ haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶.

Embodiment 21 provides the method according to any of embodiments 14-19,wherein R⁶ is hydrogen, halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, hydroxy,C₁-C₃ alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl,—C(O)—C₁-C₃ haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶.

Embodiment 22 provides the method according to any of embodiments 14-19,wherein R⁶ is hydrogen, chloro, fluoro, methyl, ethyl, difluoromethyl,hydroxy, methoxy, ethoxy, (methoxy) methyl, (ethoxy) methyl, (methoxy)ethyl, (ethoxy)ethyl, oxetanyl, tetrahydrofuranyl, —C(O)-difluoromethyl,—NH₂, or —NH(CO)CH₃.

Embodiment 23 provides the method according to any of embodiments 14-19,wherein R⁶ is halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, hydroxy, C₁-C₆alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶.

Embodiment 24 provides the method according to any of embodiments 14-19,wherein R⁶ is halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, hydroxy, C₁-C₃alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶.

Embodiment 25 provides the method according to any of embodiments 14-19,wherein R⁶ is chloro, fluoro, methyl, ethyl, difluoromethyl, hydroxy,methoxy, ethoxy, (methoxy) methyl, (ethoxy) methyl, (methoxy)ethyl,(ethoxy)ethyl, oxetanyl, tetrahydrofuranyl, —C(O)-difluoromethyl, —NH₂,or —NH(CO)CH₃.

Embodiment 26 provides the method according to any of embodiments 23-25,wherein each G, Q, J and U is independently C(H).

Embodiment 27 provides the method according to any of embodiments 23-25,wherein G, Q, J and U are independently selected from C(H) and C(R⁵).

Embodiment 28 provides the method according to any of embodiments 23-25,wherein G, Q, J and U are independently selected from C(H) and N.

Embodiment 29 provides the method according to any of embodiments 14-19,wherein

R⁶ is hydrogen;

at least one of G, Q, J, and U is C(R⁵), and the remaining G, Q, J, andU are independently selected from C(H), C(R⁵) and N, wherein each R⁵ isindependently hydroxy, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkoxy, C₁-C₆ haloalkoxy, C₃-C₆ cycloalkoxy, C₃-C₆ cycloalkyl, C₃-C₆heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl.

Embodiment 30 provides the method according to embodiment 29, whereinone or two of G, Q, J and U is N.

Embodiment 31 provides the method according to any of embodiments 14-19,wherein

R⁶ is hydrogen;

at least one of G, Q, J, and U is C(R⁵), and the remaining G, Q, J, andU are independently selected from C(H) and C(R⁵), wherein each R⁵ isindependently hydroxy, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkoxy, C₁-C₆ haloalkoxy, C₃-C₆ cycloalkoxy, C₃-C₆ cycloalkyl, C₃-C₆heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl.

Embodiment 32 provides the method according to embodiment 31, wherein atleast one of G, Q, J, and U is C(R⁵), and the remaining G, Q, J, and Uare independently C(H); for example only one of G, Q, J, and U is C(R⁵).

Embodiment 33 provides the method according to embodiment 31, whereintwo of G, Q, J, and U is C(R⁵), and the remaining G, Q, J, and U areindependently C(H).

Embodiment 34 provides the method according to embodiment 31, whereinthree of G, Q, J, and U is C(R⁵), and the remaining G, Q, J, and U isC(H).

Embodiment 35 provides the method according to any of embodiments 14-19,wherein G, Q, J, and U together with the thiophene to which they areattached form:

Embodiment 36 provides the method according to embodiment 35, wherein G,Q, J, and U together with the thiophene ring to which they are attachedform a benzo[b]thiophene.

Embodiment 37 provides the method according to any one of embodiments14-36, wherein R⁵, if present, is hydroxy, halogen, C₁-C₃ alkyl, C₁-C₃haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, C₃-C₆ cycloalkoxy, C₃-C₆cycloalkyl, C₃-C₆ heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl.

Embodiment 38 provides the method according to any one of embodiments14-36, wherein R⁵, if present, is hydroxy, halogen, C₁-C₃ alkyl, C₁-C₃haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, C₃-C₆ heterocycloalkyl, orC₁-C₃ alkoxyC₁-C₃ alkyl.

Embodiment 39 provides the method according to any one of embodiments14-36, wherein R⁵, if present, is hydroxy, chloro, fluoro, methyl,ethyl, methoxy, ethoxy, 2,2-difluoroethoxy, oxetanyl, tetrahydrofuranyl,(methoxy) methyl, (ethoxy)methyl, (methoxy)ethyl, or (ethoxy)ethyl.

Embodiment 40 provides the method according to any one of embodiments14-39, wherein R⁷ is methyl.

Embodiment 41 provides the method according to any one of embodiments14-39, wherein R⁷ is ethyl.

Embodiment 42 provides the method according to any one of embodiments14-39, wherein R⁷ is propyl (e.g., isopropyl).

Embodiment 43 provides the method according to any one of embodiments14-39, wherein R⁷ is difluoromethyl or trifluoromethyl.

Embodiment 44 provides the method according to embodiment 14, whereinthe PRMT5 inhibitor is of the formula:

wherein

G, Q, J, and U together with the thiophene to which they are attachedform:

-   -   where each R⁵ is independently hydroxy, halogen, C₁-C₃ alkyl,        C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, C₃-C₆        heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl; and

R⁶ is hydrogen, halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, hydroxy, C₁-C₃alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶.

Embodiment 45 provides the method according to embodiment 14, whereinthe PRMT5 inhibitor is of the formula:

wherein

G, Q, J, and U together with the thiophene to which they are attachedform:

-   -   where each R⁵ is independently hydroxy, halogen, C₁-C₃ alkyl,        C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, C₃-C₆        heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl; and

R⁶ is halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, hydroxy, C₁-C₃ alkoxy,C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃ haloalkyl,—N(R⁹)₂, or —NR¹⁵(CO)R¹⁶.

Embodiment 46 provides the method according to embodiment 14, whereinthe

PRMT5 inhibitor is of the formula:

wherein

G, Q, J, and U together with the thiophene to which they are attachedform:

-   -   where each R⁵ is independently hydroxy, halogen, C₁-C₃ alkyl,        C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, C₃-C₆        heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl.

Embodiment 47 provides the method of the disclosure wherein the PRMT5inhibotor is a compound of the formula (IIIB):

or a pharmaceutically acceptable salt thereof, wherein

A is CR⁹ or N;

D is —CH₂—NH₂,

W is CR⁹ or N, where R⁹ is H or C₁-C₃ alkyl;R⁵¹ is hydrogen, fluoro, chloro, or methyl, or R⁵¹ and R⁵² together withatoms to which they are attached form a C₄-C₆ heterocycloalkyl (e.g,hydrofuranyl);R⁵² is fluoro, chloro, or methyl, or R⁵² and R⁵³ together with atoms towhich they are attached form a phenyl;R⁵³ is hydrogen, fluoro, chloro, or methyl;R⁵⁴ is hydrogen, halogen, C₁-C₃ alkyl, or C₁-C₃ alkoxy;L⁵ is —O— or —CH₂—;R⁶ is hydrogen, halogen, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, C₁-C₃alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃ haloalkyl, or—NR¹⁵(CO)R¹⁶, where R¹⁵ is hydrogen or methyl, and R¹⁶ is C₁-C₃ alkyl;R⁷ is C₁-C₃ alkyl or C₁-C₃ haloalkyl.

Embodiment 48 provides the method according to embodiment 47, wherein:

A is —CH or —CCH₃;

D is —CH₂—NH₂;

W is —CH, —CCH₃, or N;

R⁵¹, R⁵², R⁵³, and R⁵⁴ are each independently selected from hydrogen,fluoro, chloro, or methyl;

L⁵ is —O—;

R⁶ is hydrogen, fluoro, chloro, or methyl; and

R⁷ is C₁-C₂ alkyl or C₁-C₂ haloalkyl.

Embodiment 49 provides the method according to embodiment 47 orembodiment 48, wherein:

A and W are —CH;

D is —CH₂—NH₂;

R⁵¹, R⁵², and R⁵³ are each independently selected from hydrogen, fluoro,chloro, and methyl;

R⁵⁴ is hydrogen;

L⁵ is —O—;

R⁶ is hydrogen; and

R⁷ is methyl.

Embodiment 50 provides the method according to any of embodiments 47-49,wherein:

A and W are —CH;

D is —CH₂—NH₂;

R⁵¹ and R⁵² are each independently selected from fluoro, chloro, andmethyl;

R⁵³ and R⁵⁴ are hydrogen;

L⁵ is —O—;

R⁶ is hydrogen; and

R⁷ is methyl.

Embodiment 51 provides the method according to embodiment 47, wherein Ais CH.

Embodiment 52 provides the method according to embodiment 47 or 48,wherein W is N.

Embodiment 53 provides the method according to embodiment 47 or 48,wherein W is CH.

Embodiment 54 provides the method according to any of embodiments 47-50,wherein D is —CH₂—NH₂.

Embodiment 55 provides the method according to any of embodiments 47-51,wherein R⁵⁴ is hydrogen or methyl.

Embodiment 56 provides the method according to any of embodiments 47-51,wherein R⁵⁴ is hydrogen.

Embodiment 57 provides the method according to any of embodiments 47-51,wherein R⁵⁴ is methyl.

Embodiment 58 provides the method according to embodiment 47, where thePRMT5 inhibitor is of the formula:

such as e.g.,

Embodiment 59 provides the method according to any of embodiments 47-55,wherein L⁵ is —CH₂—.

Embodiment 60 provides the method according to any of embodiments 47-55,wherein L⁵ is —O—.

Embodiment 61 provides the method according to any of embodiments 47-57,wherein R⁶ is hydrogen, halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, hydroxy,C₁-C₃ alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl,—C(O)—C₁-C₃ haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶; for example, wherein R⁶is hydrogen, chloro, fluoro, methyl, ethyl, difluoromethyl, hydroxy,methoxy, ethoxy, (methoxy)methyl, (ethoxy) methyl, (methoxy)ethyl,(ethoxy)ethyl, oxetanyl, tetrahydrofuranyl, —C(O)-difluoromethyl, —NH₂,or —NH(CO)CH₃.

Embodiment 62 provides the method according to any of embodiments 47-57,wherein R⁶ is hydrogen, halogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy; forexample, R⁶ is hydrogen, halogen, C₁-C₃ alkyl, or C₁-C₃ alkoxy.

Embodiment 63 provides the method according to any of embodiments 47-57,wherein R⁶ is hydrogen, chloro, fluoro, methyl, ethyl, methoxy, orethoxy.

Embodiment 64 provides the method according to any of embodiments 47-57,wherein R⁶ is halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, hydroxy, C₁-C₃alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶; for example, wherein R⁶ is chloro,fluoro, methyl, ethyl, difluoromethyl, hydroxy, methoxy, ethoxy,(methoxy) methyl, (ethoxy) methyl, (methoxy) ethyl, (ethoxy)ethyl,oxetanyl, tetrahydrofuranyl, —C(O)-difluoromethyl, —NH₂, or —NH(CO)CH₃.

Embodiment 65 provides the method according to any of embodiments 47-57,wherein R⁶ is halogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy; for example, R⁶ ishalogen, C₁-C₃ alkyl, or C₁-C₃ alkoxy.

Embodiment 66 provides the method according to any of embodiments 47-57,wherein R⁶ is chloro, fluoro, methyl, ethyl, methoxy, or ethoxy.

Embodiment 67 provides the method according to any one of embodiments47-63, wherein R⁷ is methyl.

Embodiment 68 provides the method according to any one of embodiments47-63, wherein R⁷ is ethyl.

Embodiment 69 provides the method according to any one of embodiments47-63, wherein R⁷ is propyl (e.g., isopropyl).

Embodiment 70 provides the method according to any one of embodiments47-63, wherein R⁷ is difluoromethyl or trifluoromethyl.

Embodiment 71 provides the method according to any of embodiments 47-67,wherein R⁵³ is hydrogen or methoxy; or wherein R⁵³ is hydrogen.

Embodiment 72 provides the method according to embodiment 47, where thePRMT5 inhibitor is of the formula:

Embodiment 73 provides the method according to any one of embodiments47-69, wherein R⁵² is fluoro, and R⁵¹ is hydrogen, fluoro, chloro, ormethyl.

Embodiment 74 provides the method according to any one of embodiments47-69, wherein R⁵² is fluoro, and R⁵¹ is chloro.

Embodiment 75 provides the method according to any one of embodiments47-69, wherein R⁵² is fluoro, and R⁵¹ is methyl or hydrogen (forexample, R⁵² is fluoro and R⁵¹ is methyl; or R⁵² is fluoro and R⁵¹ ishydrogen).

Embodiment 76 provides the method according to any one of embodiments47-69, wherein R⁵¹ and R⁵² together with atoms to which they areattached form a hydrofuranyl

Embodiment 77 provides the method according to any one of embodiments47-76, wherein the PRMT5 inhibitor is

Embodiment 78 provides the method according to any one of embodiments47-77, wherein the PRMT5 inhibitor is

One aspect of the disclosure provides the method wherein the PRMT5inhibitor is a compound of the formula (IIIA) (Embodiment 79):

or a pharmaceutically acceptable salt thereof, wherein

A is CR⁹ or N;

D is —CH₂—NH₂,

W is CR⁹ or N, where R⁹ is H or C₁-C₃ alkyl;

R² is

where R⁵⁶ is hydrogen, fluoro, chloro, or methyl,

G, Q, J and U are independently selected from C(H), C(R⁵), and N,provided only one or two of G, Q, J, and U can be N;

each R⁵ is independently hydroxy, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₃-C₆ cycloalkoxy, C₃-C₆ cycloalkyl,C₃-C₆ heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl;

R⁶ is hydrogen, halogen, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, C₁-C₃alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃ haloalkyl, or—NR¹⁵(CO)R¹⁶, where R¹⁵ is hydrogen or methyl, and R¹⁶ is C₁-C₃ alkyl;andR⁷ is C₁-C₃ alkyl or C₁-C₃ haloalkyl.

One aspect of the disclosure provides the method wherein the PRMT5inhibitor is a compound of the formula (IIIA) (Embodiment 80):

or a pharmaceutically acceptable salt thereof, wherein

A is CR⁹ or N;

D is —CH₂—NH₂,

W is CR⁹ or N, where R⁹ is H or C₁-C₃ alkyl;

R² is

where R⁵⁶ is hydrogen, halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkoxy, or C₁-C₆ haloalkoxy;

R⁶ is hydrogen, halogen, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, C₁-C₃alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃ haloalkyl, or—NR¹⁵(CO)R¹⁶, where R¹⁵ is hydrogen or methyl, and R¹⁶ is C₁-C₃ alkyl;andR⁷ is C₁-C₃ alkyl or C₁-C₃ haloalkyl.

Embodiment 81 provides the method according to embodiment 79 or 80,wherein A is CH.

Embodiment 82 provides the method according to embodiment 79 or 80,wherein W is N.

Embodiment 83 provides the method according to embodiment 79 or 80,wherein W is CH.

Embodiment 84 provides the method according to any of embodiments 79 or80, wherein D is —CH₂—NH₂.

Embodiment 85 provides the method according to embodiment 79 or 80,which is of the formula:

Embodiment 86 provides the method according to embodiment 79 or 81-85,wherein R² is

Embodiment 87 provides the method according to embodiment 86, wherein G,Q, J and U are independently selected from C(H) and C(R⁵).

Embodiment 88 provides the method according to embodiment 86, wherein G,Q, J and U are independently C(H).

Embodiment 89 provides the method according to embodiment 86, wherein atleast one of G, Q, J, and U is C(R⁵), and the remaining G, Q, J, and Uare independently C(H); for example only one of G, Q, J, and U is C(R⁵).

Embodiment 90 provides the method according to embodiment 86, wherein Uis N, and G, Q, and J are independently selected from C(H) and C(R⁵).

Embodiment 91 provides the method according to embodiment 86, wherein Gis N, and Q, J, and U are independently selected from C(H) and C(R⁵).

Embodiment 92 provides the method according to any one of embodiments 79or 81-91, wherein R⁵, if present, is hydroxy, halogen, C₁-C₃ alkyl,C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, C₃-C₆ cycloalkoxy,C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, or C₁-C₃ alkoxyC₁-C₃ alkyl.

Embodiment 93 provides the method according to any one of embodiments 79or 81-91, wherein R⁵, if present, is hydroxy, halogen, C₁-C₃ alkyl,C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, C₃-C₆ heterocycloalkyl,or C₁-C₃ alkoxyC₁-C₃ alkyl.

Embodiment 94 provides the method according to any one of embodiments 79or 81-91, wherein R⁵, if present, is hydroxy, chloro, fluoro, methyl,ethyl, methoxy, ethoxy, 2,2-difluoroethoxy, oxetanyl, tetrahydrofuranyl,(methoxy) methyl, (ethoxy)methyl, (methoxy)ethyl, or (ethoxy)ethyl.

Embodiment 95 provides the method according to any one of embodiments 79or 81-91, wherein R⁵, if present, is halogen, C₁-C₆ alkyl, or C₁-C₆alkoxy; for example, R⁶ is halogen, C₁-C₃ alkyl, or C₁-C₃ alkoxy.

Embodiment 96 provides the method according to any one of embodiments 79or 81-91, wherein R⁵, if present, is chloro, fluoro, methyl, ethyl,methoxy, or ethoxy.

Embodiment 97 provides the method according to any one of embodiments 79or 81-91, wherein R⁵⁶ is fluoro, chloro, or methyl.

Embodiment 98 provides the method according to embodiment 80-85, whereinR² is

Embodiment 99 provides the method according to any of embodiments 80-85or 98, wherein R⁵⁶ is hydrogen, fluoro, chloro, or methyl.

Embodiment 100 provides the method according to any of embodiments79-99, wherein R⁶ is hydrogen, halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl,hydroxy, C₁-C₃ alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl,—C(O)—C₁-C₃ haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶; for example, wherein R⁶is hydrogen, chloro, fluoro, methyl, ethyl, difluoromethyl, hydroxy,methoxy, ethoxy, (methoxy)methyl, (ethoxy) methyl, (methoxy)ethyl,(ethoxy)ethyl, oxetanyl, tetrahydrofuranyl, —C(O)-difluoromethyl, —NH₂,or —NH(CO)CH₃.

Embodiment 101 provides the method according to any of embodiments79-99, wherein R⁶ is hydrogen, halogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy;for example, R⁶ is hydrogen, halogen, C₁-C₃ alkyl, or C₁-C₃ alkoxy.

Embodiment 102 provides the method according to any of embodiments79-99, wherein R⁶ is hydrogen, chloro, fluoro, methyl, ethyl, methoxy,or ethoxy.

Embodiment 103 provides the method according to any of embodiments79-99, wherein R⁶ is halogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, hydroxy,C₁-C₃ alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl,—C(O)—C₁-C₃ haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶; for example, wherein R⁶is chloro, fluoro, methyl, ethyl, difluoromethyl, hydroxy, methoxy,ethoxy, (methoxy) methyl, (ethoxy) methyl, (methoxy) ethyl,(ethoxy)ethyl, oxetanyl, tetrahydrofuranyl, —C(O)-difluoromethyl, —NH₂,or —NH(CO)CH₃.

Embodiment 104 provides the method according to any of embodiments79-99, wherein R⁶ is halogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy; for example,R⁶ is halogen, C₁-C₃ alkyl, or C₁-C₃ alkoxy.

Embodiment 105 provides the method according to any of embodiments79-99, wherein R⁶ is chloro, fluoro, methyl, ethyl, methoxy, or ethoxy.

Embodiment 106 provides the method according to any one of embodiments79-105, wherein R⁷ is methyl.

Embodiment 107 provides the method according to any one of embodiments79-105, wherein R⁷ is ethyl.

Embodiment 108 provides the method according to any one of embodiments79-105, wherein R⁷ is propyl (e.g., isopropyl).

Embodiment 109 provides the method according to any one of embodiments79-105, wherein R⁷ is difluoromethyl or trifluoromethyl.

In certain embodiments of the methods of the disclosure as describedherein, the PRMT5 inhibitor is:

In certain embodiments of the methods of the disclosure as describedherein, the PRMT5 inhibitor is:

In certain embodiments of the methods of the disclosure as describedherein, the PRMT5 inhibitor is:

In certain embodiments of the methods of the disclosure as describedherein, the PRMT5 inhibitor is:

The PRMT5 inhibitor of the disclosure and/or the CDK4/6 inhibitor of thedisclosure may be provided as a pharmaceutical composition comprising atherapeutically effective amount of such inhibitor and apharmaceutically acceptable carrier, excipient, and/or diluents. ThePRMT5 inhibitor of the disclosure and/or the CDK4/6 inhibitor of thedisclosure may be formulated by any method well known in the art and maybe prepared for administration by any route, including, withoutlimitation, parenteral, oral, sublingual, transdermal, topical,intranasal, intratracheal, or intrarectal. In certain embodiments, ThePRMT5 inhibitor of the disclosure and/or the CDK4/6 inhibitor of thedisclosure are administered intravenously in a hospital setting. Incertain other embodiments, administration may preferably be by the oralroute.

The characteristics of the carrier will depend on the route ofadministration. As used herein, the term “pharmaceutically acceptable”means a non-toxic material that is compatible with a biological systemsuch as a cell, cell culture, tissue, or organism, and that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s). Thus, pharmaceutical compositions of thedisclosure may contain, in addition to the inhibitor, diluents, fillers,salts, buffers, stabilizers, solubilizers, and other materials wellknown in the art. The preparation of pharmaceutically acceptableformulations is described in, e.g., Remington's Pharmaceutical Sciences,18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.

The PRMT5 inhibitor and the CDK4/6 inhibitor of the disclosure areadministered in a therapeutically effective amount. As used herein, thephrase “therapeutically effective amount” or “effective amount” refersto the amount of active agent that elicits the biological or medicinalresponse that is being sought in a tissue, system, subject or human by aresearcher, medical doctor or other clinician. In general, thetherapeutically effective amount is sufficient to deliver the biologicalor medicinal response to the subject without causing serious toxiceffects. A dose of the active agent may be in the range from about 0.01to 300 mg/kg per day, such as 0.1 to 100 mg/kg per day, more generally0.5 to about 25 mg/kg body weight of the recipient per day. A typicaltopical dosage will range from 0.01 to 3% wt/wt in a suitable carrier.

In certain embodiments of the methods of the disclosure, thetherapeutically effective amount of the PRMT5 inhibitor is in the rangeof about 0.01 to 300 mg/kg per day. For example, in certain embodiments,the therapeutically effective amount of the PRMT5 inhibitor is in therange of about 0.1 to 100 mg/kg per day, or 25 to 100 mg/kg per day, or50 to 100 mg/kg per day.

In certain embodiments, the therapeutically effective amount of thePRMT5 inhibitor is less than 1% of, e.g., less than 10%, or less than25%, or less than 50% of the clinically-established therapeutic amount(e.g., such as the amount required when the PRMT5 inhibitor isadministered by itself).

In certain embodiments of the methods of the disclosure, thetherapeutically effective amount of the CDK4/6 inhibitor is in the rangeof about 0.01 to 300 mg/kg per day. For example, in certain embodiments,the therapeutically effective amount of the CDK4/6 inhibitor is in therange of about 0.1 to 100 mg/kg per day, or 0.1 to 50 mg/kg per day, or10 to 100 mg/kg per day, or 10 to 50 mg/kg per day.

In certain embodiments, the therapeutically effective amount of theCDK4/6 inhibitor is less than 1% of, e.g., less than 10%, or less than25%, or less than 50% of the clinically-established therapeutic amount(e.g., such as the amount required when the CDK4/6 inhibitor isadministered by itself).

Combination therapy, in defining use of PRMT5 inhibitor and the CDK4/6inhibitor of the present disclosure, is intended to embraceadministration of each agent in a sequential manner in a regimen thatwill provide beneficial effects of the drug combination (e.g., the PRMT5inhibitor and the CDK4/6 inhibitor of the disclosure can be formulatedas separate compositions that are given sequentially), and is intendedas well to embrace co-administration of these agents in a substantiallysimultaneous manner, such as in a single dosage form having a fixedratio of these active agents or in multiple or a separate dosage formsfor each agent. The disclosure is not limited in the sequence ofadministration: the PRMT5 inhibitor of the disclosure may beadministered either prior to or after (i.e., sequentially), or at thesame time (i.e., simultaneously) as administration of the CDK4/6inhibitor of the disclosure.

The methods of disclosure are useful as a first-line treatment. Thus, incertain embodiments of the methods of the disclosure, the subject hasnot previously received another first-line of therapy.

The methods of disclosure are also useful as a first-line maintenance ora second-line treatment. Thus, in certain embodiments of the methods ofthe disclosure, the subject has previously completed another first-lineof therapy. For example, the methods of the disclosure, in certainembodiments, may provide a delay in progression and relapse of cancer insubjects that have previously completed another first-line chemotherapy.For example, in certain embodiments, the subject has previouslycompleted a platinum- and/or taxane-based chemotherapy (e.g.,carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, and thelike). In certain embodiments of the methods of the disclosure, thesubject has previously completed another first-line chemotherapy and isin partial response to such chemotherapy.

Definitions

For simplicity, chemical moieties are defined and referred to throughoutprimarily as univalent chemical moieties (e.g., alkyl, aryl, etc.).Nevertheless, such terms may also be used to convey correspondingmultivalent moieties under the appropriate structural circumstancesclear to those skilled in the art. For example, while an “alkyl” moietygenerally refers to a monovalent radical (e.g. CH₃—CH₂—), in certaincircumstances a bivalent linking moiety can be “alkyl,” in which casethose skilled in the art will understand the alkyl to be a divalentradical (e.g., —CH₂—CH₂—), which is equivalent to the term “alkylene.”(Similarly, in circumstances in which a divalent moiety is required andis stated as being “aryl,” those skilled in the art will understand thatthe term “aryl” refers to the corresponding divalent moiety, arylene.)All atoms are understood to have their normal number of valences forbond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 forS, depending on the oxidation state of the S).

The term “amino” refers to —NH₂.

The term “acetyl” refers to “—C(O)CH₃.

As herein employed, the term “acyl” refers to an alkylcarbonyl orarylcarbonyl substituent wherein the alkyl and aryl portions are asdefined herein.

The term “alkyl” as employed herein refers to saturated straight andbranched chain aliphatic groups having from 1 to 12 carbon atoms. Assuch, “alkyl” encompasses C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁and C₁₂ groups. Examples of alkyl groups include, without limitation,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, and hexyl.

The term “alkenyl” as used herein means an unsaturated straight orbranched chain aliphatic group with one or more carbon-carbon doublebonds, having from 2 to 12 carbon atoms. As such, “alkenyl” encompassesC₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁ and C₁₂ groups. Examples ofalkenyl groups include, without limitation, ethenyl, propenyl, butenyl,pentenyl, and hexenyl.

The term “alkynyl” as used herein means an unsaturated straight orbranched chain aliphatic group with one or more carbon-carbon triplebonds, having from 2 to 12 carbon atoms. As such, “alkynyl” encompassesC₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁ and C₁₂ groups. Examples ofalkynyl groups include, without limitation, ethynyl, propynyl, butynyl,pentynyl, and hexynyl.

An “alkylene,” “alkenylene,” or “alkynylene” group is an alkyl, alkenyl,or alkynyl group, as defined hereinabove, that is positioned between andserves to connect two other chemical groups. Examples of alkylene groupsinclude, without limitation, methylene, ethylene, propylene, andbutylene. Exemplary alkenylene groups include, without limitation,ethenylene, propenylene, and butenylene. Exemplary alkynylene groupsinclude, without limitation, ethynylene, propynylene, and butynylene.

The term “alkoxy” refers to —OC₁-C₆ alkyl.

The term “cycloalkyl” as employed herein is a saturated and partiallyunsaturated cyclic hydrocarbon group having 3 to 12 carbons. As such,“cycloalkyl” includes C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁ and C₁₂cyclic hydrocarbon groups. Examples of cycloalkyl groups include,without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroalkyl” refers to an alkyl group, as defined hereinabove,wherein one or more carbon atoms in the chain are independently replacedO, S, or NRX, wherein Rx is hydrogen or C₁-C₃ alkyl. Examples ofheteroalkyl groups include methoxymethyl, methoxyethyl andmethoxypropyl.

An “aryl” group is a C₆-C₁₄ aromatic moiety comprising one to threearomatic rings. As such, “aryl” includes C₆, C₁₀, C₁₃, and C₁₄ cyclichydrocarbon groups. An exemplary aryl group is a C₆-C₁₀ aryl group.Particular aryl groups include, without limitation, phenyl, naphthyl,anthracenyl, and fluorenyl. An “aryl” group also includes fusedmulticyclic (e.g., bicyclic) ring systems in which one or more of thefused rings is non-aromatic, provided that at least one ring isaromatic, such as indenyl.

An “aralkyl” or “arylalkyl” group comprises an aryl group covalentlylinked to an alkyl group wherein the moiety is linked to another groupvia the alkyl moiety. An exemplary aralkyl group is—(C₁-C₆)alkyl(C₆-C₁₀)aryl, including, without limitation, benzyl,phenethyl, and naphthylmethyl. For example, an arC₁-C₃alkyl is an arylgroup covalently linked to a C₁-C₃ alkyl.

A “heterocyclyl” or “heterocyclic” group is a mono- or bicyclic (fusedor spiro) ring structure having from 3 to 12 atoms, (3, 4, 5, 6, 7, 8,9, 10, 11 or 12 atoms), for example 4 to 8 atoms, wherein one or morering atoms are independently —C(O)—, N, NR⁴, O, or S, and the remainderof the ring atoms are quaternary or carbonyl carbons. Examples ofheterocyclic groups include, without limitation, epoxy, oxiranyl,oxetanyl, azetidinyl, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, piperazinyl,imidazolidinyl, thiazolidinyl, thiatanyl, dithianyl, trithianyl,azathianyl, oxathianyl, dioxolanyl, oxazolidinyl, oxazolidinonyl,decahydroquinolinyl, piperidonyl, 4-piperidonyl, thiomorpholinyl,dimethyl-morpholinyl, and morpholinyl. Specifically excluded from thescope of this term are compounds having adjacent ring O and/or S atoms.

As used herein, “L-heterocyclyl” refers to a heterocyclyl groupcovalently linked to another group via an alkylene linker.

As used herein, the term “heteroaryl” refers to a group having 5 to 14ring atoms, preferably 5, 6, 10, 13 or 14 ring atoms; having 6, 10, or14π electrons shared in a cyclic array; and having, in addition tocarbon atoms, from one to three heteroatoms that are each independentlyN, O, or S. Heteroaryl also includes fused multicyclic (e.g., bicyclic)ring systems in which one or more of the fused rings is non-aromatic,provided that at least one ring is aromatic and at least one ringcontains an N, O, or S ring atom. Examples of heteroaryl groups includeacridinyl, azocinyl, benzimidazolyl, benzofuranyl,benzo[d]oxazol-2(3H)-one, 2H-benzo[b][1,4]oxazin-3(4H)-one,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, furanyl, furazanyl, imidazolinyl, imidazolyl,1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolinyl,2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl,quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, andxanthenyl.

A “L-heteroaralkyl” or “L-heteroarylalkyl” group comprises a heteroarylgroup covalently linked to another group via an alkylene linker.Examples of heteroalkyl groups comprise a C₁-C₆ alkyl group and aheteroaryl group having 5, 6, 9, or 10 ring atoms. Examples ofheteroaralkyl groups include pyridylmethyl, pyridylethyl,pyrrolylmethyl, pyrrolylethyl, imidazolylmethyl, imidazolylethyl,thiazolylmethyl, thiazolylethyl, benzimidazolylmethyl,benzimidazolylethyl quinazolinylmethyl, quinolinylmethyl,quinolinylethyl, benzofuranylmethyl, indolinylethyl isoquinolinylmethyl,isoinodylmethyl, cinnolinylmethyl, and benzothiophenylethyl.Specifically excluded from the scope of this term are compounds havingadjacent ring O and/or S atoms.

An “arylene,” “heteroarylene,” or “heterocyclylene” group is a bivalentaryl, heteroaryl, or heterocyclyl group, respectively, as definedhereinabove, that is positioned between and serves to connect two otherchemical groups.

As employed herein, when a moiety (e.g., cycloalkyl, aryl, heteroaryl,heterocyclyl, urea, etc.) is described as “optionally substituted”without expressly stating the substituents it is meant that the groupoptionally has from one to four, preferably from one to three, morepreferably one or two, non-hydrogen substituents.

The term “halogen” or “halo” as employed herein refers to chlorine,bromine, fluorine, or iodine.

The term “haloalkyl” refers to an alkyl chain in which one or morehydrogens have been replaced by a halogen. Exemplary haloalkyls aretrifluoromethyl, difluoromethyl, flurochloromethyl, chloromethyl, andfluoromethyl.

The term “hydroxyalkyl” refers to -alkylene-OH.

EXAMPLE

The methods of the disclosure are illustrated further by the followingexamples, which is not to be construed as limiting the disclosure inscope or spirit to the specific procedures and compounds described inthem.

Study Design

The PRMT5 inhibitors of the disclosure demonstrate selective activity inMTAP-deleted cancers by binding to and further inhibiting PRMT5 whenbound to the intracellular metabolite MTA. As noted above, MTAP is anenzyme in the methionine salvage pathway and its deletion in cancercells leads to the accumulation of MTA in these cells. PRMT5 is anessential enzyme required for cell viability and, as such, the PRMT5inhibitors of the disclosure represent a novel approach to selectivelytreat MTAP-deleted cancers.

A single mutation will likely not cause cancer—most often, it ismultiple mutations that are responsible for developing cancer. Theinventors found the treatment of certain cancers with PRMT5 inhibitorsimproved with the use of combination therapies. Particularly, theinventors surprisingly found that a combination therapy of PRMT5inhibitor and CDK4/6 inhibitor provides greater antitumor activitycompared to either inhibitor alone.

Study Procedure

Immunodeficient female nu/nu mice were implanted with 5×10⁶ LU99 lungcancer cells in 50% Matrigel. Tumors were measured using calipers untilthey reached approximately 150-200 mm³. Animals were randomized toreceive A) vehicle (0.5% methylcellulose (4000 cps)/0.2% Tween80 inwater), B) a PRMT5 inhibitor, C) CDK4/6 inhibitor, or D) the PRMT5inhibitor and CDK4/6 inhibitor, all administered orally (PO) for 21days. Tumor volume was measured twice a week (n=5/treatment group).Average tumor volume and standard error of the mean was calculated andplotted at each study day in GraphPad.

Example 1

This example was carried out according to the study procedure describedabove. The PRMT5 inhibitor was MRTX9768 administered at 100 mg/kg twicea day (BID). MRTX9768 is2-(4-(4-(aminomethyl)-1-oxo-1,2-dihydrophthalazin-6-yl)-1-methyl-1H-pyrazol-5-yl)-3-fluoro-1-naphthonitrile,disclosed as Example 16-1 at p. 304 of the International patentpublication No. WO 2021/050915 A1, published 18 Mar. 2021, incorporatedby reference in its entirety.

The CDK4/6 inhibitor used in this example was palbociclib administeredat 130 mg/kg once a day (QD). Palbociclib is6-acetyl-8-cyclopentyl-5-methyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrido[2,3-d]pyrimidin-7-one,and is sold as Ibrance® available from Pfizer Inc., New York, N.Y.

Results are provided in FIG. 1 and Table 1. The combination of MRTX9768and MRTX849 led to greater antitumor activity compared to eitherinhibitor alone in this KRAS^(G12C) and CDKN2A/MTAP^(DEL) lung tumorxenograft LU99 model.

TABLE 1 Tumor Volume (mm³) Group Day 0 3 7 10 14 17 21 Vehicle Mean181.04 276.67 467.48 718.83 1040.93 1358.61 1951.06 (PO QD) SEM 18.8429.58 61.05 103.76 215.23 266.16 306.05 MRTX9768 Mean 183.59 265.26331.80 349.53 406.58 431.91 458.95 (100 mg/kg PO BID) SEM 15.07 16.0724.14 25.13 43.52 51.50 60.11 palbociclib Mean 184.39 247.02 355.77482.60 731.56 914.41 1148.44 (130 mg/kg PO QD) SEM 15.37 29.94 47.9758.36 93.09 114.17 133.46 MRTX9768 Mean 184.20 262.26 287.14 309.96335.73 335.87 298.87 (100 mg/kg PO BID) + SEM 15.75 20.21 24.40 27.6524.97 27.15 39.04 palbociclib (130 mg/kg PO QD)

Example 2

This example was carried out according to the study procedure describedabove. The PRMT5 inhibitor was MRTX7477, administered at 200 mg/kg BID.MRTX7477 is24444-(aminomethyl)-1-oxo-1,2-dihydrophthalazin-6-yl)-1-methyl-1H-pyrazol-5-yl)-1-naphthonitrile,disclosed as Example 4-147 at p. 226 of the International patentpublication No. WO 2021/050915 A1, published 18 Mar. 2021, incorporatedby reference in its entirety. The CDK4/6 inhibitor used in this examplewas the same as in Example 1, palbociclib, administered at 130 mg/kg QD.

Results are provided in FIG. 2 and Table 2. The combination of MRTX7477and MRTX849 led to greater antitumor activity compared to eitherinhibitor alone in this KRAS^(G12C) and CDKN2A/MTAP^(DEL) lung tumorxenograft LU99 model.

TABLE 2 Tumor Volume (mm³) Group −2 1 5 8 12 15 19 21 Vehicle 126.3230.8 372.8 511.0 724.4 981.3 1357.6 1669.9 (PO QD) MRTX7477 129.4 214.5210.7 177.1 185.3 189.1 229.5 267.5 (200 mg/kg PO BID) Palbociclib 133.4209.8 215.9 277.3 359.4 413.5 675.9 827.0 (130 mg/kg PO QD) MRTX7477135.3 203.1 165.8 159.4 138.5 122.8 123.2 127.7 (200 mg/kg PO BID) +palbociclib (130 mg/kg PO QD)

Examples 3 to 6

The compound of the disclosure was evaluated in several different lungtumor xenograft models: HCC4006, SW1573 PRMT5-044, H1650, and A549PRMT-034. This example was carried out substantially according to thestudy procedure described above, except with mice bearing HCC4006xenograft tumors, SW1573 PRMT5-044 xenograft tumors, H1650 xenografttumors, or A549 PRMT-034 xenograft tumors. The PRMT5 inhibitor wasMRTX1719, administered at 50 mg/kg QD or at 100 mg/kg QD. MRTX1719 is(2M)-2-(4-(4-(aminomethyl)-1-oxo-1,2-dihydrophthalazin-6-yl)-1-methyl-1H-pyrazol-5-yl)-4-chloro-6-cyclopropoxy-3-fluorobenzonitrile,disclosed as Example 16-8 at p. 307 of the International patentpublication No. WO 2021/050915 A1, published 18 Mar. 2021, incorporatedby reference in its entirety. The CDK4/6 inhibitor used in this examplewas the same as in Example 1, palbociclib, administered at 130 mg/kg QD.

The results for HCC4006 are provided in FIG. 3 and Table 3.

TABLE 3 Tumor Volume (mm³) Group Day 0 2 5 9 12 16 20 Vehicle Mean 126168 188 209 248 278 297 (PO QD) SEM 6 9 16 15 18 15 14 MRTX1719 Mean 126150 159 154 156 159 78 (100 mg/kg PO QD) SEM 7 12 12 13 10 11 7Palbociclib Mean 127 155 143 117 115 111 100 (130 mg/kg PO QD) SEM 7 107 7 8 5 6 MRTX1719 Mean 127 180 178 141 142 128 102 (100 mg/kg PO QD) +SEM 8 28 29 25 27 18 18 Palbociclib (130 mg/kg PO QD)

The results for SW1573 PRMT5-044 are provided in FIG. 4 and Table 4.

TABLE 4 Tumor Volume (mm³) Grroup Day 0 3 8 10 14 17 21 Vehicle Mean 145182 273 355 471 590 727 (PO QD) SEM 12 9 27 42 57 87 121 MRTX1719 Mean138 163 259 324 375 437 547 (50 mg/kg PO QD) SEM 9 19 40 46 55 72 117Palbociclib Mean 140 160 208 260 293 302 319 (130 mg/kg PO QD) SEM 9 1017 20 25 28 40 MRTX1719 Mean 143 162 170 189 223 238 251 (50 mg/kg POQD) + SEM 10 18 25 33 34 44 48 Palbociclib (130 mg/kg PO QD)

The results for H1650 are provided in FIG. 5 and Table 5.

TABLE 5 Tumor Volume (mm³) Grroup Day 0 3 7 10 14 17 20 Vehicle Mean 147264 473 685 971 1210 1361 (PO QD) SEM 12 19 54 58 97 96 131 MRTX1719Mean 146 231 471 599 625 711 635 (100 mg/kg PO QD) SEM 11 15 38 66 80104 107 Palbociclib Mean 147 268 356 493 480 589 743 (130 mg/kg PO QD)SEM 11 37 48 61 67 97 90 MRTX1719 Mean 147 204 307 398 322 296 271 (100mg/kg PO QD) + SEM 8 22 37 38 26 24 21 Palbociclib (130 mg/kg PO QD)

The results for A549 PRMT-034 are provided in FIG. 6 and Table 6.

TABLE 6 Tumor Volume (mm³) Group Day 1 5 8 12 15 19 22 26 29 34 VehicleMean 119 157 214 240 300 354 378 506 567 726 (PO QD) SEM 9 18 31 37 5271 78 124 156 271 MRTX1719 Mean 118 169 200 210 214 221 263 288 325 (100mg/kg PO SEM 9 15 24 29 34 36 38 49 52 57 QD) Palbociclib Mean 118 134155 177 189 179 186 220 255 263 (130 mg/kg PO SEM 8 10 10 17 22 19 21 3247 48 QD) MRTX1719 Mean 119 144 166 180 183 182 194 199 206 220 (100mg/kg PO SEM 9 11 24 28 29 30 38 42 43 49 QD) + Palbociclib (130 mg/kgPO QD)

Examples 7 and 8

The compound of the disclosure was evaluated in a couple of differentpancreatic tumor xenograft models: PANC-05-04 and BXPC-3. This examplewas carried out substantially according to the study procedure describedabove, except with mice bearing PANC-05-04 xenograft tumors or BXPC-3xenograft tumors. The PRMT5 inhibitor and the CDK4/6 inhibitor used inthis example were the same as in Example 3: MRTX1719 was administered at100 mg/kg QD, and palbociclib was administered at 130 mg/kg QD.

The results for PANC-05-04 are provided in FIG. 7 and Table 7.

TABLE 7 Tumor Volume (mm³) Group Day 0 3 7 10 14 17 20 Vehicle Mean 150335 447 534 654 757 832 (PO QD) SEM 10 50 95 86 121 141 176 MRTX1719Mean 149 382 519 559 563 624 603 (100 mg/kg PO QD) SEM 9 63 58 87 88 73119 Palbociclib Mean 149 319 365 378 341 391 350 (130 mg/kg PO QD) SEM 738 39 37 28 39 49 MRTX1719 Mean 150 296 360 348 347 334 312 (100 mg/kgPO QD) + SEM 7 19 70 114 120 124 96 Palbociclib (130 mg/kg PO QD)

The results for BXPC-3 are provided in FIG. 8 and Table 8.

TABLE 8 Tumor Volume (mm³) Group Day 0 3 6 9 13 16 20 Vehicle Mean 122202 277 331 410 479 536 (PO QD) SEM 9 25 36 60 77 99 112 MRTX1719 Mean122 174 206 310 359 372 368 (50 mg/kg PO QD) SEM 10 9 8 29 55 63 49Palbociclib Mean 122 162 174 187 195 190 193 (130 mg/kg PO QD) SEM 11 1015 17 27 23 38 MRTX1719 Mean 121 141 137 111 86 102 122 (50 mg/kg POQD) + SEM 8 13 13 15 9 4 23 Palbociclib (130 mg/kg PO QD)

Example 9

The compound of the disclosure was evaluated in a gastric tumorxenograft model MKN45, and the results are provided in FIG. 9 and Table9. This example was carried out substantially according to the studyprocedure described above, except with mice bearing MKN45 xenografttumors. The PRMT5 inhibitor and the CDK4/6 inhibitor used in thisexample were the same as in Example 3: MRTX1719 was administered at 100mg/kg QD, and palbociclib was administered at 130 mg/kg QD.

TABLE 9 Tumor Volume (mm³) Grroup Day 0 3 7 10 14 17 20 Vehicle Mean 106180 297 388 535 763 801 (PO QD) SEM 15 31 48 58 66 69 71 MRTX1719 Mean105 165 207 221 214 230 221 (100 mg/kg PO QD) SEM 13 27 38 43 37 50 55Palbociclib Mean 106 158 236 264 330 434 474 (130 mg/kg PO QD) SEM 13 1936 34 27 52 64 MRTX1719 Mean 105 155 190 178 199 187 182 (100 mg/kg POQD) + SEM 13 18 20 17 25 17 26 Palbociclib (130 mg/kg PO QD)

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be incorporated within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated herein by referencefor all purposes.

What is claimed is:
 1. A method for treating cancer in a subject, themethod comprising: administering to the subject a therapeuticallyeffective amount of a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitorand a therapeutically effective amount of a protein arginine N-methyltransferase 5 (PRMT5) inhibitor.
 2. The method of claim 1, wherein thecancer comprises methylthioadenosine phosphorylase (MTAP) genehomozygous deletion.
 3. The method of claim 1, wherein the cancercomprises a cyclin-dependent kinase inhibitor 2A (CDKN2A) genehomozygous deletion.
 4. The method of claim 2, wherein the cancerfurther comprises a Kirsten rat sarcoma viral oncogene homologglycine-to-cysteine (KRAS^(G12C)) gene mutation.
 5. The method of claim1, wherein the cancer is lung cancer, pancreatic cancer, head and neckcancer, bladder cancer, esophageal cancer, lymphoma, stomach cancer,skin cancer, breast cancer, brain cancer, liver cancer, and coloncancer.
 6. The method of claim 1, wherein the cancer is lung cancer(e.g., mesothelioma or non-small cell lung cancer (NSCLC) includingadenocarcinoma and squamous cell), pancreatic cancer, head and neckcancer, bladder cancer, esophageal cancer, lymphoma (e.g., diffuse largeB-cell lymphoma), stomach cancer, melanoma, breast cancer, and braincancer (e.g., glioblastoma multiforme and glioma).
 7. The method ofclaim 1, wherein the cancer is lung cancer, such as NSCLC ormesothelioma.
 8. The method of claim 1, wherein the CDK4/6 inhibitor isCDK2/4/6.
 9. The method of claim 1, wherein the CDK4/6 inhibitor isselected from palbociclib, abemaciclib, ribociclib, PF-06873600, andcombinations thereof.
 10. The method of claim 1, wherein the CDK4/6inhibitor is palbociclib.
 11. The method of claim 1, wherein the PRMT5inhibitor is a methylthioadenosine (MTA)-cooperative PRMT5 inhibitor.12. The method of claim 1, wherein the PRMT5 inhibitor is a compound ofFormula IIA, IIB or IIC:

or a pharmaceutically acceptable salt thereof, wherein: A is CR⁹ or N; Dis (C(R⁹)₂)₁₋₂—NH₂,

or D is

where the methylene is bonded to E where E is C; E is C, CR⁹ or N; eachL is independently a bond or C₁-C₃ alkylene; W is CR⁹ or N; each X isindependently a bond, O, S, —NR⁴— or —NR⁴C(O)—; each Z is independentlya bond, —SO—, —SO₂—, —CH(OH)— or —C(O)—; each R² is independentlyhydroxy, halogen, cyano, cyanomethyl, —(NR⁴)₂, hydroxyalkyl, alkoxy,—SO₂C₁-C₃alkyl, —X-arC₁-C₃alkyl, heteroalkyl, C₂-C₄ alkynyl,—X-haloalkyl, —X—C₁-C₅ alkyl, —Z—C₁-C₅ alkyl, heterocyclyl,—X-L-cycloalkyl, —Z-cycloalkyl, —X-aryl, —Z-aryl, or —X-heteroaryl,wherein the heterocyclyl, the cycloalkyl, the aryl and the heteroarylare optionally substituted with one or more R⁵; each R⁴ is independentlyhydrogen or C₁-C₃ alkyl; each R⁵ is independently cyano, oxo, halogen,C₁-C₃ alkyl, hydroxyalkyl, hydroxy, alkoxy, alkoxy-C₁-C₃ alkyl,—X-haloalkyl, —Z-cycloalkyl, —X-arC₁-C₃alkyl, —X-arC₁-C₃alkylsubstituted with cyano, —X-L-cycloalkyl optionally substituted withC₁-C₃ alkyl or oxo, —X-L-heteroaryl optionally substituted with one ormore C₁-C₃ alkyl or oxo, —X-L-heterocyclyl optionally substituted withone or more C₁-C₃ alkyl or oxo, or -X-aryl; R⁶ is hydrogen, halogen,C₁-C₃ alkyl, haloalkyl, hydroxy, alkoxy, C₁-C₃ alkyl-alkoxy, N(R⁹)₂,NR⁹C(O)R⁹, C(O)R⁹, oxetane and THF; R⁷ is H or C₁-C₃ alkyl optionallysubstituted with one or more halogen; R⁸ is H or C₁-C₃ alkyl; and eachR⁹ is independently H or C₁-C₃ alkyl, halogen or haloalkyl.
 13. Themethod of claim 1, wherein the PRMT5 inhibitor is a compound of FormulaIIIA:

or a pharmaceutically acceptable salt thereof, wherein A is CR⁹ or N; Dis —CH₂—NH₂,

W is CR⁹ or N, where R⁹ is H or C₁-C₃ alkyl; R² is

 where R⁵⁶ is hydrogen, fluoro, chloro, or methyl,  G, Q, J and U areindependently selected from C(H), C(R⁵), and N, provided only one or twoof G, Q, J, and U can be N; each R⁵ is independently hydroxy, halogen,C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₃-C₆cycloalkoxy, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, or C₁-C₃alkoxyC₁-C₃ alkyl; R⁶ is hydrogen, halogen, C₁-C₆ alkyl, hydroxy, C₁-C₆alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃haloalkyl, or —NR¹⁵(CO)R¹⁶, where R¹⁵ is hydrogen or methyl, and R¹⁶ isC₁-C₃ alkyl; and R⁷ is C₁-C₃ alkyl or C₁-C₃ haloalkyl.
 14. The method ofclaim 13, wherein the PRMT5 inhibitor is:

or a pharmaceutically acceptable salt thereof.
 15. The method of claim1, wherein the PRMT5 inhibitor is a compound of Formula IIIB:

or a pharmaceutically acceptable salt thereof, wherein A is CR⁹ or N; Dis —CH₂—NH₂,

W is CR⁹ or N, where R⁹ is H or C₁-C₃ alkyl; R⁵¹ is hydrogen, fluoro,chloro, or methyl, or R⁵¹ and R⁵² together with atoms to which they areattached form a C₄-C₆ heterocycloalkyl (e.g, hydrofuranyl); R⁵² isfluoro, chloro, or methyl, or R⁵² and R⁵³ together with atoms to whichthey are attached form a phenyl; R⁵³ is hydrogen, fluoro, chloro, ormethyl; R⁵⁴ is hydrogen, halogen, C₁-C₃ alkyl, or C₁-C₃ alkoxy; L⁵ is—O— or —CH₂—; R⁶ is hydrogen, halogen, C₁-C₆ alkyl, hydroxy, C₁-C₆alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆ heterocycloalkyl, —C(O)—C₁-C₃haloalkyl, or —NR¹⁵(CO)R¹⁶, where R¹⁵ is hydrogen or methyl, and R¹⁶ isC₁-C₃ alkyl; R⁷ is C₁-C₃ alkyl or C₁-C₃ haloalkyl.
 16. The method ofclaim 15, wherein: A is —CH or —CCH₃; D is —CH₂—NH₂; W is —CH, —CCH₃, orN; R⁵¹, R⁵², R⁵³, and R⁵⁴ are each independently selected from hydrogen,fluoro, chloro, or methyl; L⁵ is —O—; R⁶ is hydrogen, fluoro, chloro, ormethyl; and R⁷ is C₁-C₂ alkyl or C₁-C₂ haloalkyl.
 17. The method ofclaim 15, wherein: A and W are —CH; D is —CH₂—NH₂; R⁵¹, R⁵², and R⁵³ areeach independently selected from hydrogen, fluoro, chloro, and methyl;R⁵⁴ is hydrogen; L⁵ is —O—; R⁶ is hydrogen; and R⁷ is methyl.
 18. Themethod of claim 1, wherein: A and W are —CH; D is —CH₂—NH₂; R⁵¹ and R⁵²are each independently selected from fluoro, chloro, and methyl; R⁵³ andR⁵⁴ are hydrogen; L⁵ is —O—; R⁶ is hydrogen; and R⁷ is methyl.
 19. Themethod of claim 15, wherein the PRMT5 inhibitor is:

or a pharmaceutically acceptable salt thereof.
 20. The method of claim15, wherein the PRMT5 inhibitor is:

or a pharmaceutically acceptable salt thereof.
 21. The method of claim1, wherein the PRMT5 inhibitor is MRTX1719 or a pharmaceuticallyacceptable salt thereof, and the CDK4/6 inhibitor is palbociclib. 22.The method of claim 1, wherein the PRMT5 inhibitor is a compound ofFormula IIIC:

or a pharmaceutically acceptable salt thereof, wherein A is CR⁹ or N; Dis —CH₂ —NH₂,

W is CR⁹ or N, where R⁹ is H or C₁-c₃ alkyl; G, Q, J and U areindependently selected from C(H), C(R⁵), and N, provided only one or twoof G, Q, J, and U can be N; each R⁵ is independently hydroxy, halogen,C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₃-C₆cycloalkoxy, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, or C₁-C₃alkoxyC₁-C₃ alkyl; R⁶ is hydrogen, halogen, C₁-C₆ alkyl, C₁-C₆haloalkyl, hydroxy, C₁-C₆ alkoxy, C₁-C₃ alkoxyC₁-C₃ alkyl, C₃-C₆heterocycloalkyl, —C(O)—C₁-C₃ haloalkyl, —N(R⁹)₂, or —NR¹⁵(CO)R¹⁶, whereeach R⁹ is independently H or C₁-C₃ alkyl, R¹⁵ is hydrogen or methyl,and R¹⁶ is C₁-C₃ alkyl; and R⁷ is C₁-C₃ alkyl or C₁-C₃ haloalkyl. 23.The method of claim 22, wherein the PRMT5 inhibitor is:

or a pharmaceutically acceptable salt thereof.
 24. The method of claim1, wherein the therapeutically effective amount of the PRMT5 inhibitoris in the range of about 0.01 to 300 mg/kg per day.
 25. The method ofclaim 1, wherein the therapeutically effective amount of the PRMT5inhibitor is in the range of about 0.1 to 100 mg/kg per day.
 26. Themethod of claim 1, wherein the therapeutically effective amount of thePRMT5 inhibitor is less than 1% of, e.g., less than 10%, or less than25%, or less than 50% of the clinically-established therapeutic amount.27. The method of claim 1, wherein the therapeutically effective amountof the CDK4/6 inhibitor is in the range of about 0.01 to 300 mg/kg perday.
 28. The method of claim 1, wherein the therapeutically effectiveamount of the CDK4/6 inhibitor is in the range of about 0.1 to 100 mg/kgper day.
 29. The method of claim 1, wherein the therapeuticallyeffective amount of the CDK4/6 inhibitor is less than 1% of, e.g., lessthan 10%, or less than 25%, or less than 50% of theclinically-established therapeutic amount.
 30. The method of claim 1,wherein the CDK4/6 inhibitor and the PRMT5 inhibitor are administeredsequentially.
 31. The method of claim 1, wherein the CDK4/6 inhibitorand the PRMT5 inhibitor are administered simultaneously.
 32. The methodof claim 1, wherein the subject previously received or completed afirst-line chemotherapy.
 33. The method of claim 32, wherein thefirst-line chemotherapy is platinum- and/or taxane-based chemotherapy.